US6677596B2 - Method and apparatus for the detection of fluorescent light in confocal scanning microscopy - Google Patents

Method and apparatus for the detection of fluorescent light in confocal scanning microscopy Download PDF

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Publication number
US6677596B2
US6677596B2 US09/948,297 US94829701A US6677596B2 US 6677596 B2 US6677596 B2 US 6677596B2 US 94829701 A US94829701 A US 94829701A US 6677596 B2 US6677596 B2 US 6677596B2
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light
light source
fluorescent
illuminating
specimen
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US20020027202A1 (en
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Johann Engelhardt
Juergen Hoffmann
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Leica Microsystems CMS GmbH
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Leica Microsystems Heidelberg GmbH
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/008—Details of detection or image processing, including general computer control
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes

Definitions

  • the present invention concerns a method for the detection of fluorescent light in scanning microscopy. Moreover, the invention concerns an apparatus for the detection of fluorescent light.
  • an apparatus for the detection of fluorescent light comprising:
  • the fluorescent photon yield of the fluorescent light of the fluorescent excitation induced by means of multi-photon excitation processes depends on several influencing variables.
  • the properties of the light source causing the multi-photon excitation, the system parameters of the confocal scanning microscope, and the properties of the fluorescing materials are critical. Only when all the influences affecting the fluorescent photon yield are matched and adapted to one another can optimized fluorescent excitation and fluorescent light detection be accomplished.
  • the lifetime of the excited states of the fluorescing materials is provided for as a possible property of the fluorescing materials to which the operating parameters of the light source causing the multi-photon excitation and/or the system parameters of the confocal microscope could be adapted. Also provided for is an adaptation to the effective cross section of the excitation of the fluorescing materials, to the excitation and/or emission wavelength, and/or an adaptation to the bleaching behavior of the fluorescing materials. These are thus the most important properties of the fluorescing materials to which the light source causing the multi-photon excitation, and the system parameters of the confocal scanning microscope, are to be adapted.
  • a plurality of variant methods which will be discussed in more detail below, are provided for modifying the operating parameters of the light source causing the multi-photon excitation.
  • the output of the light source could be correspondingly adjusted.
  • the output of the light source would need to be made greater than or equal to the output which corresponds to the saturation output of the fluorescing materials.
  • the saturation output of the fluorescing materials depends on the illumination pattern used for multi-photon excitation of the fluorescing materials, since with increasing illumination volume, the light output to be introduced into the confocal scanning microscope correspondingly increases.
  • the pulse duration of the light emitted by the light source could moreover be adjusted correspondingly.
  • a prechirp unit such as is known, for example, from DE 44 37 896 C1 could be used.
  • this prechirp unit in particular, light pulses that have experienced a pulse widening as a result of interactions with optical components such as, e.g. glass fibers or lenses, are compressed back to their original pulse duration.
  • This prechirp unit could, however, in particularly advantageous fashion, be used to influence the pulse duration of the light emitted by the light source, in order to optimize the fluorescent photon yield of the fluorescing materials.
  • the pulse repetition rate of the light emitted by the light source is to be correspondingly adjusted.
  • the pulse repetition rate of the light emitted from the light source is to be adjusted, or optionally varied, as a function of the lifetime of the excited states of the fluorescing materials and on the basis of their saturation behavior. Pulse repetition rates of 75 to 100 MHz are usually used for two-photon excitations of fluorescing materials; the use of pulse repetition rates in ranges from kHz to GHz can be useful for optimum fluorescent photon yield.
  • a modification of the pulse repetition rate could be achieved, in the context of mode-coupled light sources, by the fact that the resonator length of the light source is modified.
  • a decrease in the resonator length would yield an increase in the pulse repetition rate; this can be achieved, for example, with a titanium:sapphire laser.
  • a resonator length in the centimeter range would yield a pulse repetition rate of a few GHz.
  • a corresponding increase in the resonator length would correspondingly lower the pulse repetition rate.
  • a multiplication of the pulse repetition rate, in the context of actively mode-coupled light sources could be achieved by having several pulses circulate simultaneously in the resonator.
  • the active mode coupling control system would need to be set appropriately so as thereby to achieve, for example, an eight-fold increase in the pulse repetition rate.
  • a pulsed laser with Q-switching could be used as the light source.
  • generation of the light pulses is achieved by a corresponding Q-switching system, which in turn can be modified over a specific range so that the pulse repetition rate of this type of light source can be adapted to the excitation properties of the fluorescing materials by varying the Q-switching system as well.
  • mode-coupled laser systems could be used as the light source; in particular, titanium:sapphire lasers could be utilized. If the light output necessary for optimum excitation of the fluorescing materials is greater than the light output that mode-coupled laser systems usually provide, a regenerative amplifier could be arranged downstream from the mode-coupled laser system.
  • a semiconductor laser could also be used as the light source for multi-photon excitation of fluorescing materials.
  • This semiconductor laser could be caused to pulse by gain switching.
  • the pulse repetition rate of the semiconductor laser can thereby be adjusted from the Hz range into the GHz range.
  • An alternative light source could be a flash lamp-pumped laser system, which substantially exhibits pulse repetition rates that substantially correspond to the frequency of the flash lamp.
  • an OPO optical parametric oscillator
  • OPA optical parametric amplifier
  • the dynamic range of the detector system and/or on the other hand the pixel integration time of the detector system could be correspondingly adjusted.
  • the amplification bandwidth of the detected signals and the scanning rate of the beam deflection apparatus of the confocal scanning microscope are also correlated herewith, in particular, the amplification bandwidth of the detected signals and the scanning rate of the beam deflection apparatus of the confocal scanning microscope.
  • a planar detector could be used for detection of the specimen points excited by an arrangement of this kind.
  • multi-photon excitation one or more confocal detection pinholes can be dispensed with; the reason is that multi-photon excitation can occur only in the focal region of the microscope objective, since only there is the excitation output of the light source high enough.
  • illumination with multiple focal points provision could be made for illumination with a focal line, for example using a slit-shaped illumination and detection system. In this context, however, the resolution capability is confocal only perpendicular to the focal line; the resolution existing along the focal line is the same as that available in conventional microscopy.
  • Fluorescent dyes, caged compounds, and/or nanocrystals could be used as fluorescing materials. These are fluorescing materials that are commonly used in the biomedical field and in the field of biological and biochemical basic research; biological specimens can be specifically bound to the fluorescing materials using, for example, antibody bonds to complementary specimen regions.
  • FIG. 1 schematically depicts a first exemplary embodiment of an apparatus according to the present invention.
  • FIG. 2 shows a diagram in which the fluorescent photon yield is plotted as a function of the repetition rate.
  • the operating parameters of light source 2 causing the multi-photon excitation, and the system parameters of the confocal scanning microscope are adapted to the properties of the fluorescing materials.
  • the pulse repetition rate of light source 2 is defined according to the diagram of FIG. 2 .
  • the fluorescent photon yield is plotted as a function of pulse repetition rate, for a constant average light output of light source 2 .
  • the fluorescent photon yield is plotted on the diagram as a bold solid line.
  • the fluorescent photon yield cannot be arbitrarily increased at a lower repetition rate and thus at a higher pulse output (the average light output of light source 2 being constant), and the maximum attainable fluorescent photon yield is therefore plotted as a dashed line.
  • Light source 2 comprises a titanium:sapphife laser 3 and a regenerative amplifier 4 downstream from titanium:sapphire laser 3 .
  • the light emitted from light source 2 is directed, after reflection at mirror 5 , onto beam divider 6 .
  • Beam divider 6 divides illuminating light beam 7 into three partial illuminating beams 8 .
  • Scanning mirror 9 mounted tiltably and configured as a dichroic beam splitter, reflects the three partial illuminating beams 8 and deflects them as a result of the tilting, so that specimen 1 can be scanned by the deflection of scanning mirror 9 .
  • the deflection of scanning mirror 9 is accomplished in two directions substantially perpendicular to one another, only one of which is drawn.
  • the fluorescing materials used for two-photon excitation are fluorescent dyes that are specifically bound to individual specimen regions of specimen 1 .

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Engineering & Computer Science (AREA)
  • Computer Vision & Pattern Recognition (AREA)
  • General Engineering & Computer Science (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
US09/948,297 2000-09-07 2001-09-06 Method and apparatus for the detection of fluorescent light in confocal scanning microscopy Expired - Lifetime US6677596B2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10044308A DE10044308A1 (de) 2000-09-07 2000-09-07 Verfahren und Vorrichtung zur Detektion von Fluoreszenzlicht bei der konfokalen Rastermikroskopie
DE10044308 2000-09-07
DEDE10044308.7 2000-09-07

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EP (1) EP1186882B1 (de)
JP (1) JP5076204B2 (de)
DE (1) DE10044308A1 (de)

Cited By (7)

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Publication number Priority date Publication date Assignee Title
US20050259319A1 (en) * 2004-05-19 2005-11-24 Gary Brooker Method and system for wide-field multi-photon microscopy having a confocal excitation plane
US20060140467A1 (en) * 2004-12-28 2006-06-29 Olympus Corporation Image processing apparatus
US20080116392A1 (en) * 2006-11-20 2008-05-22 Celloptic, Inc. Method and system for wide-field multi-photon microscopy having a confocal excitation plane
US20100118549A1 (en) * 2008-11-07 2010-05-13 Endure Medical, Inc. Stereoscopic Illumination System for Microscope
US20120182413A1 (en) * 2011-01-19 2012-07-19 California Institute Of Technology Plane-projection multi-photon microscopy
US9846121B2 (en) 2009-06-17 2017-12-19 W.O.M. World Of Medicine Gmbh Device and method for multi-photon fluorescence microscopy for obtaining information from biological tissue
DE102017125688A1 (de) 2017-11-03 2019-05-09 Leica Microsystems Cms Gmbh Verfahren und Vorrichtung zum Abrastern einer Probe

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US6859273B2 (en) * 2001-07-23 2005-02-22 University Of Rochester Method for operating a laser scanning confocal microscope system and a system thereof
FR2857107B1 (fr) * 2003-03-20 2008-04-04 Vincent Lauer Dispositif optique confocal avec changement de miroir
JP4629658B2 (ja) * 2003-03-20 2011-02-09 ヴァンサン・ロウエ ミラーの交換を伴う共焦点装置
DE10313988B4 (de) * 2003-03-27 2014-03-27 Leica Microsystems Cms Gmbh Verfahren zur Prüfung der Qualität von Mikroskopen
JP2005321453A (ja) * 2004-05-06 2005-11-17 Olympus Corp 顕微鏡用蛍光照明装置
US20090218514A1 (en) * 2004-12-10 2009-09-03 Koninklijke Philips Electronics, N.V. Multi-spot investigation apparatus
HU227859B1 (en) * 2005-01-27 2012-05-02 E Szilveszter Vizi Real-time 3d nonlinear microscope measuring system and its application
WO2006085894A1 (en) * 2005-01-31 2006-08-17 Chemimage Corporation Apparatus and method for chemical imaging of a biological sample
DE102005027896B4 (de) * 2005-06-16 2012-03-15 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Verfahren zum optischen Messen einer Probe
WO2008010120A2 (en) * 2006-07-17 2008-01-24 Koninklijke Philips Electronics N.V. Employing beam scanning for optical detection
DE102007052551B4 (de) 2007-10-30 2019-06-19 Carl Zeiss Microscopy Gmbh Verfahren zur Durchführung einer Rasterbildkorrelationsspektroskopiemessung sowie Steuereinheit, Laser-Scanning-Mikroskop und Computerprogramm
JP5203063B2 (ja) * 2008-06-24 2013-06-05 オリンパス株式会社 多光子励起測定装置
KR101802462B1 (ko) * 2016-04-21 2017-11-28 서울대학교산학협력단 각도의존성 광발광 측정장치
KR20180077898A (ko) * 2016-12-29 2018-07-09 엘지디스플레이 주식회사 검사장치 및 이를 이용한 검사방법
EP4143544A4 (de) * 2020-04-30 2024-05-22 Univ Michigan Regents Gleichzeitige detektion von laseremission und fluoreszenz
DE102022112384B4 (de) 2022-05-17 2024-02-29 Abberior Instruments Gmbh Verfahren, lichtmikroskop und computerprogramm zum einstellen einer zeitverzögerung zwischen lichtpulsen

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Cited By (16)

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Publication number Priority date Publication date Assignee Title
US20050259319A1 (en) * 2004-05-19 2005-11-24 Gary Brooker Method and system for wide-field multi-photon microscopy having a confocal excitation plane
US7170675B2 (en) 2004-05-19 2007-01-30 Celloptic, Inc. Method and system for wide-field multi-photon microscopy having a confocal excitation plane
US20070091307A1 (en) * 2004-05-19 2007-04-26 Celloptic, Inc. Method and system for wide-field multi-photon microscopy having a confocal excitation plane
US7468837B2 (en) 2004-05-19 2008-12-23 Celloptic, Inc. Wide-field multi-photon microscope having simultaneous confocal imaging over at least two pixels
US20060140467A1 (en) * 2004-12-28 2006-06-29 Olympus Corporation Image processing apparatus
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US20080116392A1 (en) * 2006-11-20 2008-05-22 Celloptic, Inc. Method and system for wide-field multi-photon microscopy having a confocal excitation plane
WO2010053748A1 (en) * 2008-11-07 2010-05-14 Endure Medical Inc. Stereoscopic illumination system for microscope
US20100118549A1 (en) * 2008-11-07 2010-05-13 Endure Medical, Inc. Stereoscopic Illumination System for Microscope
US8177394B2 (en) 2008-11-07 2012-05-15 Endure Medical, Inc. Stereoscopic illumination system for microscope
US9846121B2 (en) 2009-06-17 2017-12-19 W.O.M. World Of Medicine Gmbh Device and method for multi-photon fluorescence microscopy for obtaining information from biological tissue
US20120182413A1 (en) * 2011-01-19 2012-07-19 California Institute Of Technology Plane-projection multi-photon microscopy
US9494781B2 (en) * 2011-01-19 2016-11-15 California Institute Of Technology Plane-projection multi-photon microscopy
DE102017125688A1 (de) 2017-11-03 2019-05-09 Leica Microsystems Cms Gmbh Verfahren und Vorrichtung zum Abrastern einer Probe
WO2019086680A1 (de) 2017-11-03 2019-05-09 Leica Microsystems Cms Gmbh Verfahren und vorrichtung zum abrastern einer probe
US11747604B2 (en) 2017-11-03 2023-09-05 Leica Microsystems Cms Gmbh Method and device for scanning a sample

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EP1186882B1 (de) 2016-11-16
EP1186882A2 (de) 2002-03-13
US20020027202A1 (en) 2002-03-07
JP5076204B2 (ja) 2012-11-21
DE10044308A1 (de) 2002-03-21
EP1186882A3 (de) 2004-01-02
JP2002107633A (ja) 2002-04-10

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